O'Flaherty J T, Nishihira J
J Immunol. 1987 Mar 15;138(6):1889-95.
In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.
与我们之前的报告（《生物化学与生物物理研究通讯》134:587，1986年）不同，我们现在发现蛋白激酶C（PKC）在受到血小板激活因子（PAF）或白三烯（LT）B4刺激的人多形核中性粒细胞（PMN）中被动员。因此，每种化合物的纳摩尔浓度都会导致PMN失去胞质中PKC特异性蛋白磷酸化活性以及佛波酯肉豆蔻酸酯（PMA）受体。伴随这些变化的是膜相关PMA受体的较小增加。二酰基甘油和PMA对PKC的作用非常相似。然而，与这些直接的PKC激活剂不同，PAF和LTB4仅引起胞质PKC的适度降低；仅作用于用细胞松弛素B预处理的PMN；在破坏的PMN中不会动员PKC，也不会在无细胞系统中激活PKC；就PAF而言，诱导的反应在30分钟内部分逆转。此外，PAF、LTB4及其几种结构类似物在与其各自对细胞LTB4或PAF受体的亲和力密切相关的浓度下动员PKC。因此，PAF和LTB4通过间接且明显由受体介导的机制起作用。四项观察结果表明，PAF和LTB4的细胞松弛素B依赖性脱颗粒作用涉及PKC。首先，PKC动员和脱颗粒发生在相同的刺激浓度下。其次，5-羟基二十碳四烯酸在由PAF引发时显著增强了PKC动员和脱颗粒；它对LTB4诱导的反应影响相对较小。第三，PAF诱导的动员（t1/2小于7秒）先于脱颗粒（t1/2约为20秒）。最后，一种PKC阻滞剂多粘菌素B在抑制对PAF、LTB4和PMA的脱颗粒反应方面同样有效。由于受刺激的PMN可能产生并利用PAF、LTB4和5-羟基二十碳四烯酸作为次级细胞内介质，我们的结果表明PKC是功能的核心且可能至关重要的调节因子。
